The biological activity of tumor necrosis factor (TNF) is one of the most pleiotropic. In vivo, some of its activities could be beneficial (e.g., tumor cell killing), while others detrimental (e.g., septic shock and cachexia). The long-term goal of this study is to dissect the multiple signal transduction pathways induced by TNF, in order to facilitate future development of useful therapeutics against TNF mediated human diseases with diminished side effects. The biological effects of TNF are induced upon its binding to the cell surface receptors. Two TNF receptors have been identified and cloned. Although both receptors bind TNF, the exact role each receptor plays in mediating diverse TNF biological activities is not clear. Since most cells express both receptors, a receptor-discriminating reagent can shed light on the molecular action of each individual receptor. Results with monoclonal antibodies against TNF receptors are not conclusive. Current data suggest that TNF-alpha binds to different receptors at different sites on the TNF molecule. Therefore, it seems possible that receptor-discriminating TNF mutant(s) can be created to provide alternative tool(s) needed to reveal the functional differences between the two TNF receptors. To test this hypothesis, we propose (1) to create single amino acid substitution TNF mutants and test the capability of each mutant to bind to each receptor individually. (2) To test the receptor binding inhibition activity of synthetic peptides derived from the sequences of TNF receptors. (3) TNF mutants of interest will be tested on various cell types with distinct TNF biological responses. (4) TNF mutants of interest and complexes of TNF and TNF-binding synthetic peptides will be crystallized and their structures determined by X-ray crystallography. Much useful information can be obtained by determining such structures which are much easier to solve than the structure of TNF/TNF receptor complexes. In essence, we will define a fine map of the receptor binding domains of TNF and initiate an attempt to correlate, at molecular level, the structure/functional relationship between the binding to individual receptor and the diverse biological activities of TNF. By doing so, we may obtain receptor-specific TNF mutant(s) or reveal subtle differences between the receptor binding domains which can be exploited to design receptor-discriminating agent to reveal the functional differences between the two receptors. These goals can be achieved by extending our recent mutational analysis of TNF receptor binding site. The initial round of mutational analysis by us and others, before the two receptors were identified, mapped the receptor binding site to include at least part of the intersubunit grooves of TNF homotrimer. In addition, our results revealed that residue Ser133 outside the groove may also form direct contact with the receptor. By computer analysis, surface residues in the vicinity of the intersubunit groove and residue Ser133 have been identified as the targets of our second round of mutational analysis in order to define a fine map of TNF receptor binding site(s). Important information regarding to the binding of TNF to each receptor and their functional implications can be obtained from this study whether the hypothesis tested true or not.